This program is aimed at understanding the mechanistic details of enzymes that catalyze oxidation reactions of a variety of substrates including hydrocarbons and amines. The hydrocarbon-oxidizing enzymes include the heme-containing cytochrome P450 enzymes, the diiron methane monooxygenase (MMO) enzymes and various mononculear, non-heme iron enzymes. The amine-oxidizing enzymes include flavin cofactor-containing enzymes and cytochrome P450 enzymes. A premise of the program is that a good understanding of the mechanisms will have medicinal benefits in attempts to control enzyme action. Several methods of study are involved. Mechanistic probe substrates are used extensively because this method is the only one to have produced quantitative kinetic information about intermediate lifetimes in P450 and MMO hydroxylation reactions. The probes are compounds that rearrange in a characteristic manner when they are converted into a specific intermediate, and, in doing so, provide information about the nature of the intermediates. In the case of radical intermediates, the rate constants for rearrangement are determined, and lifetimes of radical intermediates can be computed from product ratios. The design of the probes and measurements of the rate constants for radical rearrangements, including ultra fast rearrangements, are integral parts of the program. Other methods of study include spectroscopic techniques at low temperature. Cryoreduction studies of P450 enzymes using ESR and ENDOR spectrsocopies will be conducted in collaboration with another group. Cryooxidation studies of iron-porphyrins and P450 enzymes involve optical detection of transients produced by laser flash photolysis methods. Stopped-flow kinetic studies of MMO enzymes are also proposed. The P450 and MMO studies are performed with a range of enzymes. Probes for studies of amine oxidations by flavin cofactor enzymes are in advanced development, and studies will be initiated with the berberine bridge enzyme (BBE), an FAD-cofactor enzyme that catalyzes an oxidative cyclization reaction in alkaloid biosynthesis. Probes for mononuclear, non-heme iron enzymes are in early development, and the objective in the current research period will be the design of probes that are substrates for the enzymes of interest such as clavimanate synthase.